SUMMARY Reductions in intercellular communication occur in almost all forms of heart disease and lead to the arrhythmias of sudden cardiac death. The most intensely studied component of the intercalated disc of the working cardiomyocyte is the connexin 43 (Cx43) gap junction. The Cx43 gene, GJA1, encodes a polycistronic mRNA that undergoes internal translation events yielding truncated polypeptides capable of modulating gap junction formation and subject to altered regulation during stress such as ischemia and hypertrophy. Such internal translation within protein coding sequences of mammalian mRNAs has only recently been identified, and a substantial gap in the knowledge exists as to how this process is regulated by the cell and if manipulation of upstream regulatory pathways and associated proteins can be harnessed therapeutically to restore normal intercellular communication in diseased hearts. The objective of this application is to provide mechanistic insight into the role of altered translation initiation in pathological Cx43 gap junction regulation and remodeling. This work is significant because it will identify altered translation initiation as a common stress response in the heart and provide fundamental understanding of regulation of electrical communication through rapid alteration of the proteome by ribosomal initiation. Our central hypothesis is that dynamic regulation of the translational landscape of the cell governs intercellular communication, is vital in maintenance of proper cardiac electrical coupling, and is altered by the integrated stress response during pathological remodeling of the heart. We will test our central hypothesis with the following specific aims: AIM 1: Determine the role of altered translation initiation in gap junction regulation. The working hypothesis for this aim is that a cellular program exists at the level of translation initiation which regulates transition from a `junctional' to a `non- junctional' state. AIM 2: Delineate the regulatory elements of GJA1 mRNA critical for internal translation initiation. The working hypothesis for this aim is that GJA1 mRNA harbors sequence- and structure-based elements necessary for internal translation to occur via recruitment of specific RNA binding proteins. AIM 3: Test if modulation of translation initiation protects against pathological cardiac remodeling. Our working hypothesis for this aim is that the integrated stress response (ISR) alters translation initiation of GJA1, reducing GJA1-20k expression and limiting gap junction formation in stressed and hypertrophic myocardium. The work outlined in this proposal is significant because it will (i) provide mechanistic insight into the role of internal translation in regulation of gap junctions and intercellular communication during stress (ii) determine how this relates to pathological remodeling in hypertrophic and ischemic hearts, and (iii) test if modulation of internal translation is a viable therapeutic strategy for human heart disease.